The present invention relates generally to the field of cellular signal transduction and more specifically to the diagnosis and treatment of various diseases and conditions associated with abnormal cellular signal transduction pathways.
The present invention concerns methods for diagnosis and treatment of disorders characterized by abnormal cellular signal transduction. The following is a discussion of relevant art, none of which is admitted to be prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of tyrosine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs) and tyrosine phosphatases (TPs).
Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been implicated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentiation. In some instances, a single tyrosine kinase can inhibit, or stimulate, cell proliferation depending on the cellular environment in which it is expressed.
RTKs are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric changes that activate the intracellular kinase domains and result in the self-phosphorylation (autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues. Their intrinsic tyrosine kinase is activated upon ligand binding, thereby initiating a complex signal transduction pathway that begins with receptor autophosphorylation and culminates in the tyrosine phosphorylation of a variety of cellular substrates and ultimately in the initiation of nuclear events necessary for the overall cell response. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signaling molecules, thereby activating various signal transduction pathways.
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains (SRC homology domain 2) and SH3 domains (SRC homology domain 3). The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction.
A central feature of signal transduction (for reviews, see Posada and Cooper, Mol. Biol. Cell 3:583-392, 1992; Hardie, Symp. Soc. Exp. Biol. 44:241-255, 1990), is the reversible phosphorylation of certain proteins. Receptor phosphorylation stimulates a physical association of the activated receptor with target molecules. Some of the target molecules such as phospholipase Cxcex3 are in turn phosphorylated and activated. Such phosphorylation transmits a signal to the cytoplasm. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal transducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the Grb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras.
The secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular signal transduction include Aaronson, Science, 254:1146-1153, 1991; Schlessinger, Trends Biochem. Sci., 13:443-447, 1988; and Ullrich and Schlessinger, Cell, 61:203-212, 1990.
RTKs are important regulators of developmental processes, as reflected by the high level of tyrosine phosphorylation in the early mouse embryo, which decreases with progressing development and is low in adult animal tissues (Pasquale and Singer, Proc. Natl. Acad. Sci. USA 88:5449-5453, 1989). For example, the mouse c-kit proto-oncogene plays a key role in the migrational behavior of specific cell types in mouse development (Chabot et al., Nature 335:88-89, 1988; Geissler et al., Cell 55:185-192, 1988; Nocka et al., Genes Dev. 3:816-826, 1989).
Disruption of the platelet-derived growth factor receptor xcex1 (PDGF-Rxcex1) gene is responsible for the mouse patch mutation, which is characterized by prominent anatomical abnormalities in homozygotes (Stephenson et al., Proc. Natl. Acad. Sci. USA 88:6-10, 1991). Moreover, Flk-1, the cognate receptor for the vascular endothelial growth factor (VEGF), was shown to be a major regulator of vasculogenesis and angiogenesis (Millauer et al., Cell 72:835-846, 1993). Finally, in Drosophila, the RTK sevenless has a well established function in the control of photoreceptor cell fate (Basler and Hafen, Science 243:931-934, 1989), as does the RTK torso in the formation of terminal structures of Drosophila larva (Sprenger et al., Nature 338:478-483, 1989).
Among adult tissues, the brain contains the highest level of protein kinase activity, comparable to that found in embryonic tissues (Maher, P. A., J. Cell. Biol. 112:955-963, 1991). Members of the trk family of RTKs have well documented roles in promoting the differentiation and survival of diverse groups of neurons of the central and peripheral nervous systems (reviewed in Raffioni et al., Annu. Rev. Biochem. 62:823-850, 1993). The eck/eph RTK subfamily (Hirai et al., Science 238:1717-1720, 1987) currently comprises the largest subgroup of RTKs (Sajjadi and Pasquale, Oncogene 8:1807-1813, 1993), with most members being expressed in the developing or adult brain.
While RTKs such as eck (Lindberg and Hunter, Mol. Cell. Biol. 10:6316-6324, 1990), Hek2 (Bxc3x6hme et al., Oncogene 8:2857-2862, 1993), Cek6, Cek9, and Cek10 (Sajjadi and Pasquale, Oncogene 8:1807-1813, 1993) have been reported to be widely expressed in a variety of tissues, Elk and Cek5 transcripts have been found predominantly in the brain (Letwin et al., Oncogene 3:621-627, 1988; Pasquale et al., J. Neuroscience 12:3956-3967, 1992).
As first noted by Maisonpierre et al. (Maisonpierre et al., Oncogene 8:3277-3288, 1993), there is a subclass of RTKs within the eck/eph family which, while being strongly expressed in the brain, are also found in other tissues, especially during embryogenesis. This subfamily includes Ehk-1, Ehk-2, (Maisonpierre et al., Oncogene 8:3277-3288, 1993), Mek4, Cek4, Hek (Sajjadi et al., New Biol. 3:769-778, 1991; Wicks et al., Proc. Natl. Acad. Sci. USA 89:1611-1615, 1992), eek (Chan and Watt, Oncogene 6:1057-1061, 1991), Sek (Nieto et al., Development 116:1137-1150, 1992; Gilardi-Hebenstreit et al., Oncogene 7:2499-2506, 1992), Cek7 and Cek8 (Sajjadi and Pasquale, Oncogene 8:1807-1813, 1993), whose members are more related to each other than to either of the above-mentioned kinases.
The present invention relates to MDK1 polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. In particular, this invention relates to methods for diagnosis and treatment of a disorder, most preferably a disorder characterized by an abnormality in a signal transduction pathway, wherein the signal transduction pathway involves the interaction between a MDK1 receptor tyrosine kinase and a MDK1 binding partner.
The present invention is based upon the isolation and characterization of a new member of the sub-group of the eck/eph family of RTKs referred to above, which we have designated mouse developmental kinase 1 (MDK1). MDK1, which was found using a polymerase chain reaction (PCR) based approach, exhibits complex transcriptional regulation and is expressed in at least five different forms. Along with two variants containing amino acid deletions in the membrane-proximal extracellular domain and the juxtamembrane region, we also identified two truncated versions of MDK1 which lack the catalytic kinase domain. Although MDK1 is transcribed in a variety of tissues in early stages of development, it is found exclusively in the brain, spleen, and testes of adult mice. The neuronal expression sites characterized indicate an important role for MDK1 in the development of the nervous system.
In addition, we have determined that disruption or promotion of the interaction between a MDK1 receptor tyrosine kinase and MDK1 binding partner is useful in therapeutic procedures. Thus, we have determined that a kinase, termed MDK1, is involved in a protein-protein interaction of therapeutic importance. This interaction is associated with the basic signalling function of proteins associated with various diseases or conditions. MDK1 polypeptides are involved in various signal transduction pathways and thus the present invention provides several agents and methods useful for diagnosing, treating, and preventing various diseases or conditions associated with abnormalities in these pathways.
Thus, in a first aspect the invention features an isolated, enriched, or purified nucleic acid encoding a MDK1 polypeptide.
By xe2x80x9cisolatedxe2x80x9d in reference to nucleic acid is meant a polymer of 2 (preferably 21, more preferably 39, most preferably 75) or more nucleotides conjugated to each other, including DNA or RNA that is isolated from a natural source or that is synthesized. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it and thus is meant to distinguish from isolated chromosomes.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to nucleic acid is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other DNA or RNA present, or by a preferential increase in the amount of the specific DNA or RNA sequence, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other nucleic acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no DNA or RNA from other sources. The other source DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector such as pUC19. This term distinguishes from naturally occuring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
By xe2x80x9ca MDK1 polypeptidexe2x80x9d is meant 2 (preferably 7, more preferably 13, most preferably 25) or more contiguous amino acids set forth in the full length amino acid sequence of SEQ ID NO:2, or a functional derivative thereof as described herein. The MDK1 polypeptide can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained. Examples of partial amino acid sequences are shown in SEQ ID NOS 3 and 5.
In preferred embodiments the isolated nucleic acid comprises, consists essentially of, or consists of a nucleic acid sequence set forth in the full length nucleic acid sequence SEQ ID NO:1, a functional derivative thereof, or at least 27, 30, 35, 40 or 50 contiguous nucleotides thereof; the MDK1 polypeptide comprises, consists essentially of, or consists of at least 9, 10, 15, 20, or 30 contiguous amino acids of a MDK1 polypeptide. The nucleic acid may be isolated from a natural source by cDNA cloning or subtractive hybridization; the natural source may be blood, semen, and tissue of various organisims including eukaryotes, mammals, birds, fish, plants, gorillas, rhesus monkeys, chimpanzees and humans; and the nucleic acid may be synthesized by the triester method or by using an automated DNA synthesizer. In yet other preferred embodiments the nucleic acid is a conserved or unique region, for example those useful for the design of hybridization probes to facilitate identification and cloning of additional polypeptides, the design of PCR probes to facilitate cloning of additional polypeptides, and obtaining antibodies to polypeptide regions. Examples of partial nucleic acid sequences are shown in SEQ ID NOS 4 and 6.
By xe2x80x9cconserved nucleic acid regionsxe2x80x9d, are meant regions present on two or more nucleic acids encoding a MDK1 polypeptide, to which a particular nucleic acid sequence can hybridize to under lower stringency conditions. Examples of lower stringency conditions suitable for screening for nucleic acid encoding MDK1 polypeptides are provided in Abe, et al. J. Biol. Chem., 19:13361 (1992) (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 7 out of 20 nucleotides.
By xe2x80x9cunique nucleic acid regionxe2x80x9d is meant a sequence present in a full length nucleic acid coding for a MDK1 polypeptide that is not present in a sequence coding for any other naturally occurring polypeptide. Such regions preferably comprise 12 or 20 contiguous nucleotides present in the full length nucleic acid encoding a MDK1 polypeptide.
The invention also features a nucleic acid probe for the detection of a MDK1 polypeptide or nucleic acid encoding a MDK1 polypeptide in a sample. The nucleic acid probe contains nucleic acid that will hybridize to a sequence set forth in SEQ ID NO:1 or a functional derivative thereof.
In preferred embodiments the nucleic acid probe hybridizes to nucleic acid encoding at least 12, 27, 30, 35, 40 or 50 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2 or a functional derivitive thereof. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.
Methods for using the probes include detecting the presence or amount MDK1 RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to MDK1 RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence coding for a MDK1 polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container means having disposed therein a nucleic acid probe.
The invention also features recombinant nucleic acid, preferably in a cell or an organism. The recombinant nucleic acid may contain a sequence set forth in SEQ ID NO:1 or a functional derivative thereof and a vector or a promoter effective to initiate transcription in a host cell. The recombinant nucleic acid can alternatively contain a transcriptional initiation region functional in a cell, a sequence complimentary to an RNA sequence encoding a MDK1 polypeptide and a transcriptional termination region functional in a cell.
In another aspect the invention features an isolated, enriched, or purified MDK1 polypeptide.
By xe2x80x9cisolatedxe2x80x9d in reference to a polypeptide is meant a polymer of 2 (preferably 7, more preferably 13, most prefereably 25) or more amino acids conjugated to each other, including polypeptides that are isolated from a natural source or that are synthesized. The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino acid material naturally associated with it.
By the use of the term xe2x80x9cenrichedxe2x80x9d in reference to a polypeptide is meant that the specific amino acid sequence constitutes a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. This could be caused by a person by preferential reduction in the amount of other amino acids present, or by a preferential increase in the amount of the specific amino acid sequence of interest, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other amino acid sequences present, just that the relative amount of the sequence of interest has been significantly increased. The term significant here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other amino acids of about at least 2 fold, more preferably at least 5 to 10 fold or even more. The term also does not imply that there is no amino acid from other sources. The other source amino acid may, for example, comprise amino acid encoded by a yeast or bacterial genome, or a cloning vector such as pUC19. The term is meant to cover only those situations in which man has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that an amino acid sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to a polypeptide does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the sequence is relatively purer than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/ml). Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The substance is preferably free of contamination at a functionally significant level, for example 90%, 95%, or 99% pure.
In preferred embodiments the MDK1 polypeptide contains at least 9, 10, 15, 20, or 30 contiguous amino acids of the full-length sequence set forth in SEQ ID NO:2, or a functional derivitive thereof.
In yet another aspect the invention features an antibody (e.g., a monoclonal or polyclonal antibody) having specific binding affinity to a MDK1 polypeptide. The antibody contains a sequence of amino acids that is able to specifically bind to a MDK1 polypeptide. By xe2x80x9cspecific binding affinityxe2x80x9d is meant that the antibody binds to MDK1 polypeptides with greater affinity than it binds to other polypeptides under specified conditions.
Antibodies having specific binding affinity to a MDK1 polypeptide may be used in methods for detecting the presence and/or amount of a MDK1 polypeptide is a sample by contacting the sample with the antibody under conditions such that an immunocomplex forms and detecting the presence and/or amount of the antibody conjugated to the MDK1 polypeptide. Diagnostic kits for performing such methods may be constructed to include a first container means containing the antibody and a second container means having a conjugate of a binding partner of the antibody and a label.
In another aspect the invention features a hybridoma which produces an antibody having specific binding affinity to a MDK1 polypeptide. By xe2x80x9chybridomaxe2x80x9d is meant an immortalized cell line which is capable of secreting an antibody, for example a MDK1 antibody. In preferred embodiments the MDK1 antibody comprises a sequence of amino acids that is able to specifically bind a MDK1 polypeptide.
Another aspect of the invention features a method of detecting the presence or amount of a compound capable of binding to a MDK1 polypeptide. The method involves incubating the compound with a MDK1 polypeptide and detecting the presence or amount of the compound bound to the MDK1 polypeptide.
Thus, in another aspect, the invention features a method for treatment of an organism having a disease or condition characterized by an abnormality in a signal transduction pathway, wherein the signal transduction pathway involves the interaction between a MDK1 receptor tyrosine kinase and a MDK1 binding partner. The disorder may also be characterized by an abnormal level of interaction between MDK1 receptor tyrosine kinase and a MDK1 binding partner. The method includes disrupting or promoting that interaction (or signal) in vivo. The method also involves inhibiting or promoting the activity of the complex formed between MDK1 receptor tyrosine kinase and a MDK1 binding partner.
By xe2x80x9corganismxe2x80x9d is meant any living creature. The term includes mammals, and specifically humans. Preferred organisms include mice, as the ability to treat or diagnose mice is often predictive of the ability to function in other organisms such as humans.
By xe2x80x9cdisease or conditionxe2x80x9d is meant a state in an organism, e.g., a human, which is recognized as abnormal by members of the medical community. The disease or condition may be characterized by an abnormality in one or more signal transduction pathways in a cell, preferably a neuronal, fibroblast, epithelial, blood or cancer cell, wherein one of the components of the signal transduction pathway is a MDK1 receptor tyrosine kinase.
Examples of diseases or conditions to be treated or diagnosed by the present invention include neurodegenerative disorders, neuroproliferative disorders, cancers, hyperproliferative disorders such as psoriasis and neurofibromatosis, inflammatory disorders, Alzheimer""s disease, Parkinson""s disease, Lou Gehrig""s disease (ALS), trauma, damaged or severed nerve injuries, Huntington""s chorea, multiple sclerosis, muscular dystrophy, syringomiplia, Tabes Dorsalis, and cardiovascular accidents. These and other diseases or conditions are often characterized by one or more of the following symptoms: tumors, astasia, aphasia, paralysis, paresea, and paralagies.
By xe2x80x9cabnormalityxe2x80x9d is meant a level which is statistically different from the level observed in organisms not suffering from such a disease or condition and may be characterized as either an excess amount, intensity or duration of signal or a deficient amount, intensity or duration of signal. The abnormality in signal transduction may be realized as an abnormality in neuronal or cancer cell function, viability or differentiation state. We have determined that such abnormal interaction in a pathway can be alleviated by action at the MDK1 -binding partner interaction site in the pathway.
An abnormal interaction level may also either be greater or less than the normal level and may impair the normal performance or function of the organism. Thus, it is also possible to screen for agents that will be useful for treating a disease or condition, characterized by an abnormality in the signal transduction pathway, by testing compounds for their ability to affect the interaction between a MDK1 receptor tyrosine kinase and a MDK1 binding partner, since the complex formed by such interaction is part of the signal transduction pathway. However, the disease or condition may be characterized by an abnormality in the signal transduction pathway even if the level of interaction between MDK1 receptor tyrosine kinase and a MDK1 binding partner is normal.
By xe2x80x9cinteractxe2x80x9d is meant any physical association between proteins, whether covalent or non-covalent. Examples of non-covalent bonds include electrostatic bonds, hydrogen bonds, and Van der Waals bonds. Stryer, Biochemistry, 1988, pages 7-8. Furthermore, the interactions between proteins may either be direct or indirect. Another example of an indirect interaction is the independent production, stimulation, or inhibition of both MDK1 receptor tyrosine kinase and a MDK1 binding partner by a regulatory agent. Depending upon the type of interaction present, various. methods may be used to measure the level of interaction. For example, the strengths of covalent bonds are often measured in terms of the energy required to break a certain number of bonds (i.e., kcal/mol) Non-covalent interactions are often described as above, and also in terms of the distance between the interacting molecules. Indirect interactions may be described in a number of ways, including the number of intermediary agents involved, or the degree of control exercised over the MDK1 receptor tyrosine kinase relative to the control exercised over the MDK1 binding partner.
By xe2x80x9cMDK1 receptor tyrosine kinasexe2x80x9d is meant an amino acid sequence substantially similar to the sequence shown in FIG. 1, or fragments thereof and is specifically meant to include human equivalents of MDK1. A sequence that is substantially similar will have at least 70% identity (preferably at least 80% and most preferably 90-100%) to the sequence of FIG. 1 in the ectodomain and at least 85% identity (preferably 90%, most preferably 95-100%) in the intracellular domains.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. MDK1.T1, MDK1.T2, MDK1.xcex941 and MDK1.xcex942 are all examples of sequences with sufficient identity to the sequence of FIG. 1 to be considered a MDK1 receptor tyrosine kinase. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
By xe2x80x9cMDK1 binding partnerxe2x80x9d is meant an amino acid sequence that interacts with or binds a MDK1 RTK. The term includes ligands and/or substrates for the MDK1 kinase.
By xe2x80x9cdisruptxe2x80x9d is meant that the interaction between the MDK1 receptor tyrosine kinase and a MDK1 binding partner is reduced either by preventing expression of the MDK1 receptor tyrosine kinase, or by preventing expression of the MDK1 binding partner, or by specifically preventing interaction of the naturally synthesized proteins having these domains or by interfering with the interaction of the proteins.
By xe2x80x9cpromotexe2x80x9d is meant that the interaction between a MDK1 receptor tyrosine kinase and a MDK1 binding partner is increased either by increasing expression of a MDK1 receptor tyrosine kinase, or by increasing expression of a MDK1 binding partner, or by decreasing the dephosphorylating activity of the corresponding regulatory TP (or other phosphatase acting on other phosphorylated signalling components) by promoting interaction of the MDK1 receptor tyrosine kinase and a MDK1 binding partner or by prolonging the duration of the interaction. Many bivalent or polyvalent linking agents are useful in coupling polypeptides, such as an antibody, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984, J. Immunol. 133:1335-2549; Jansen, F. K., et al. 1982, Immunological Rev. 62:185-216; and Vitetta et al., supra).
By xe2x80x9csignal transduction pathwayxe2x80x9d is meant the sequence of events that involves the transmission of a message from an extracellular protein to the cytoplasm through a cell membrane. The signal ultimately will cause the cell to perform a particular function, for example, to uncontrollably proliferate and therefore cause cancer. Various mechanisms for the signal transduction pathway (Fry et al., Protein Science, 2:1785-1797, 1993) provide possible methods for measuring the amount or intensity of a given signal. Depending upon the particular disease associated with the abnormality in a signal transduction pathway, various symptoms may be detected. Those skilled in the art recognize those symptoms that are associated with the various other diseases described herein. Furthermore, since some adapter molecules recruit secondary signal transducer proteins towards the membrane, one measure of signal transduction is the concentration and localization of various proteins and complexes. In addition, conformational changes that are involved in the transmission of a signal may be observed using circular dichroism and fluorescence studies.
In a related aspect the invention features a method for screening for an agent useful for treatment of such a disease or condition by assaying potential agents for the ability to disrupt or promote that interaction. The screening may also involve assaying potential agents for the ability to remove or reduce the effect of an abnormality in a signal transduction pathway, wherein the signal transduction pathway contains a MDK1 receptor tyrosine kinase and a MDK1 binding partner.
By xe2x80x9cscreeningxe2x80x9d is meant investigating an organism for the presence or absence of a property. The process may include measuring or detecting various properties, including the level of signal transduction and the level of interaction between a MDK1 receptor tyrosine kinase and a MDK1 binding partner.
Useful agents for treatment of such diseases can be identified by standard screening protocols in which measurement of such interaction is determined. For example, such an agent may be a peptide which either comprises, consists of, or consists essentially of a MDK1 receptor tyrosine kinase or, alternatively, a fragment thereof.
By xe2x80x9ccomprisingxe2x80x9d it is meant including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory, and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
In preferred embodiments the screening involves looking for agonists or antagonists of a protein of interest, for example MPK1 or a MDK1 binding partner. The term agonist refers to agents that bind the protein and that maintain the activity of the protein to which they bind. An antagonist competes with the natural ligand for binding the protein, but does not maintain the activity of the protein to which it binds.
Another aspect of the invention features a method for diagnosis of such a disease or condition. The method includes detecting the level of interaction between a MDK1 receptor tyrosine kinase and a MDK1 binding partner.
By xe2x80x9cdiagnosisxe2x80x9d is meant any method of identifying a symptom normally associated with a given disease or condition. Thus, an initial diagnosis may be conclusively established as correct by the use of additional confirmatory evidence such as the presence of other symptoms. Current classification of various diseases and conditions is constantly changing as more is learned about the mechanisms causing the diseases or conditions. Thus, the detection of an important symptom, such as the detection of an abnormal level of interaction between the MDK1 receptor tyrosine kinases and binding partners for the kinases may form the basis to define and diagnose a newly named disease or condition.
For example, conventional neurological diseases are classified according to the presence of a particular set of symptoms. However, a subset of these symptoms may both be associated with an abnormality in a particular signalling pathway, such as the ras21 pathway and in the future these diseases may be reclassified as ras21 pathway diseases regardless of the particular symptoms observed.
In preferred embodiments the MDK1 receptor tyrosine kinase has conserved cysteine residues in the ectodomain and has FN III domains as shown in FIG. 1, is selected from the group consisting of MDK1.T1, MDK1.T2, MDK1.xcex941 and MDK1.xcex942 as shown in FIG. 2, has a tyrosine residue substituted for the phenylanaline residue at position 600, has a molecular weight of 114-120 kD, and has an intracellular domain with a molecular weight of 47 kD. Residues 18-538 defining the extracellular domain are one example of a fragment, as are other smaller or larger sequences. The MDK1 RTK may contain the key amino acids of the catalytic domain that are highlighted in bold italics in FIG. 1 and have a similar three dimensional structure to the sequence given in FIG. 1, but may have various substitutions, deletions, or additions at non-key residues, as long the sequence still binds the binding partner. In other preferred embodiments the agent is therapeutically effective and has an EC50 or IC50 as described below. An EC50 or IC50 of less than or equal to 5 xcexcM is preferable, and even more preferably less than or equal to 1 xcexcM, 100 nmolar, 10 nmolar, or 1 nmolar. Such lower EC50""s or IC50""s are advantageous since they allow lower concentrations of molecules to be used in vivo or in vitro for therapy or diagnosis. The discovery of molecules with such low EC50""s and IC50""s enables the design and synthesis of additional molecules having similar potency and effectiveness. In addition, the molecule may have an EC50 or IC50 less than or equal to 5 xcexcM at one or more, but not all cells chosen from the group consisting of parathyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, cell of the thick ascending limb of Henle""s loop and/or collecting duct, central nervous system cell, keratinocyte in the epidermis, parafollicular cell in the thyroid (C-cell), intestinal cell, trophoblast in the placenta, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting cell, glucagon-secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell and GI tract cell.
By xe2x80x9ctherapeutically effective amountxe2x80x9d is meant an amount of a pharmaceutical composition having a therapeutically relevant effect. A therapeutically relevant effect relieves to some extent one or more symptoms of the disease or condition in the patient; or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or condition. Generally, a therapeutically effective amount is between about 1 nmole and 1 xcexcmole of the molecule, depending on its EC50 or IC50 and on the age and size of the patient, and the disease associated with the patient.
In a further related aspect, the invention features a method of identifying the receptor tyrosine phosphatase responsible for dephosphorylating the activated MDK1 receptor, thereby regulating the MDK1 receptor signaling pathway. Novel methods of treatment of disorders (e.g., neurological disorders) can be based on modulating this phosphatase activity. Modulation of the RTP activity can be accomplished in a variety of ways including but not limited to the use of compounds or drugs that inhibit or enhance the RTP activity, antisense or ribozyme approaches that xe2x80x9cknock outxe2x80x9d the RTP activity, or gene therapy approaches to correct defects in the RTP or restore the regulated expression of the RTP. Compounds can be used that specifically modulate the activity of the controlling RTP, thereby prolonging or enhancing signal transduction mediated by the MDK1 receptor.
In another aspect the invention features a method for screening for human cells containing a MDK1 RTK or an equivalent sequence (i.e., one that performs a similar function in humans to that played by MDK1 in mice). The method involves identifying the novel RTK in human cells using techniques that are routine and standard in the art, such as those described herein for identifying MDK1 in mouse cells (e.g., cloning, Southern or Northern blot analysis, in situ hybridization, PCR amplification, etc.).
In preferred embodiments the method features screening cells involved in human neurological functions, such as nerve cells, for the presence of MDK1. The invention also features methods of screening human cells for binding partners of MDK1 RTKs and screening other organisms for MDK1 or the corresponding binding partner. In other preferred embodiments the agent is therapeutically effective and has an EC50 or IC50 as described herein.
In other aspects, the invention provides transgenic, nonhuman mammals containing a transgene encoding a MDK1 polypeptide or a gene effecting the expression of a MDK1 polypeptide. Such transgenic nonhuman mammals are particularly useful as an in vivo test system for studying the effects of introducing a MDK1 polypeptide, regulating the expression of a MDK1 polypeptide (i.e., through the introduction of additional genes, antisense nucleic acids, or ribozymes).
A xe2x80x9ctransgenic animalxe2x80x9d is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell. Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats. The transgenic DNA may encode for a human MDK1 polypeptide. Native expression in an animal may be reduced by providing an amount of anti-sense RNA or DNA effective to reduce expression of the receptor.
In another aspect, the invention describes a polypeptide comprising a recombinant MDK1 polypeptide or a unique fragment thereof. By xe2x80x9cunique fragment,xe2x80x9d is meant an amino acid sequence present in a full-length MDK1 polypeptide that is not present in any other naturally occurring polypeptide. Preferably, such a sequence comprises 6 contiguous amino acids present in the full sequence. More preferably, such a sequence comprises 12 contiguous amino acids present in the full sequence. Even more preferably, such a sequence comprises 18 contiguous amino acids present in the full sequence.
By xe2x80x9crecombinant MDK1 polypeptidexe2x80x9d is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location (e.g., present in a different cell or tissue than found in nature), purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
In another aspect, the invention describes a recombinant cell or tissue containing a purified nucleic acid coding for a MDK1 polypeptide. In such cells, the nucleic acid may be under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter. By xe2x80x9cexogenousxe2x80x9d it is meant a promoter that is not normally coupled in vivo transcriptionally to the coding sequence for the MDK1 polypeptide.
In another aspect, the invention features a MDK1 polypeptide binding agent able to bind to a MDK1 polypeptide. The binding agent is preferably a purified antibody which recognizes an epitope present on a MDK1 polypeptide. Other binding agents include molecules which bind to the MDK1 polypeptide and analogous molecules which bind to a MDK1 polypeptide.
By xe2x80x9cpurifiedxe2x80x9d in reference to an antibody is meant that the antibody is distinct from naturally occurring antibody, such as in a purified form. Preferably, the antibody is provided as a homogeneous preparation by standard techniques. Uses of antibodies to the cloned polypeptide include those to be used as therapeutics, or as diagnostic tools.
The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.